The invention relates to novel peptide derivatives, called memno peptides, obtainable by fermentation of Memnoniella echinata FH2272, DSM 13195, in a culture medium, a process for the preparation of the memno peptides, and the use of the memno peptides as pharmaceuticals, for example, for the production of a pharmaceutical for the treatment of cardiac insufficiency.
Cardiovascular disorders still rank first as causes of death in the western industrial countries. A not inconsiderable proportion of these are patients with the diagnosis of cardiac insufficiency. Cardiac insufficiency is understood as meaning the inadequate functioning of the heart. The heart is not able to produce an output corresponding to the mammal""s requirements. Cardiac insufficiency is an acute or chronic inability of the heart, under load or even at rest, to muster the blood output necessary for metabolism or to take up the venous return. It is the state of the heart wherein the compensation mechanisms, such as heart rate, stroke volume, or hypertension, no longer suffice for the maintenance of a normal cardiac output. Cardiac insufficiency has a variety of causes, for example inflammatory and degenerative myocardial (heart muscle) changes, coronary circulatory disorders and cardiac infarct. Cardiac insufficiency leads to changes in the peripheral circulation, to impairment of the respiration, the kidney function, and the electrolyte balance, and also to reduced power of the skeletal musculature, and in the end it frequently leads to death.
Cardiac insufficiency generally occurs at an advanced age. The incidence is 3 disorders per 1000 inhabitants per year in 35-64 year-olds and 10/1000/year in the age group from 65 to 94 years. Mortality increases in 75-year-olds almost by a factor of 200 compared with the age group between 35 and 44 years. The mortality rate has remained approximately constant between 1970 and 1983, as investigations in the USA showed. For the Federal Republic of Germany, the same numbers are to be assumed. More than 50% of patients die in the first five years after diagnosis. This statistical examination, in and of itself, shows the great importance of cardiac insufficiency for the population, but it also confirms the inadequate possibilities of medicinal treatment which are available to the physician today.
In view of the inadequacy of present treatments, new concepts have been developed which should lead to innovative cardiac remedies. The ability of the cardiac and skeletal muscles to contract and thus to perform mechanical work is dependent on (1) contractile structural elements (myofibrils) and (2) chemical energy (ATP) available to the myofibrils, which is converted into mechanical energy in the contraction process. Shortening of the myofibrils occurs in the contraction process. This may be initiated by motor nerve impulses, under the action of which calcium ions (Ca2+) enter into the sarcoplasmatic space from the extracellular space within a few milliseconds and the calcium depots are emptied. In myocardial insufficiency (cardiac insufficiency), the Ca2+ concentration in myofibrils may be reduced. Ca2+ ions, however, are indispensable for the activation of the contractile apparatus. If there is increased demand, Ca2+ is generally pumped into the sarcoplasmatic reticulum (SR) under catalysis of a membranous Ca2+-dependent Mg2+-ATPase: this enzyme is also called Sarco(Endo)plasmatic Reticulum Ca2+ATPase (SERCA2). According to hydropathic analysis, the Ca2+ATPase comprises ten transmembranous helices and a number of extramembranous loops. On the cytosolic side, domains for Ca2+ and ATP binding, for phosphorylation and for interaction with the modulator protein phospholamban (PLB) are formed. The latter is a protein pentamer, which is localized in the membrane of the SR and exerts an inhibitory influence on SERCA2 in the unphosphorylated state. Under physiological stress, a phosphorylation of PLB takes place, which increases the Ca2+ affinity of SERCA2a and thus increases the transport rate for Ca2+ ions in the SR. The phosphorylation of PLB (a 52 amino acid protein) takes place on two amino acid residues: serine-16 may be phosphorylated by the cAMP-dependent protein kinase and threonine may be phosphorylated in position 17 by the Ca2+/calmodulin-dependent kinase. This phosphorylation causes a change in confirmation in PLB followed by an increased affinity of SERCA2 for Ca2+. Anti-PLB antibodies are able to imitate the PLB phosphorylation effect and thus confirm the key role of PLB as a regulator of the contractile activity of the heart (Phospholamban: Protein Structure, Mechanism of Action and Role in Cardiac Function. H. K. Simmerman and L. R. Jones, Physiological Reviews; Vol. 78, No. 4, 921ff, 1998). Activators of SERCA2 should thus bring about a favorable influence in cardiac insufficiency.
It has surprisingly been found that cultures of the fungal strain Memnoniellla echinata FH 2272, DSM 13195 contain natural substances which are able to display favorable effects on the heart and the circulation. The isolated active compounds, the memno peptides, are natural substances comprising specific constituent groups. These consitiuent groups include terpene units, a so-called polyketide moiety and a nitrogen-containing group.
Terpenes are naturally occurring compounds which can be interpreted formally as polymerization products of the hydrocarbon isoprene. According to the number of isoprene groups, monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20) etc. can be differentiated. A large number of compounds can be formed from the parent structures by substitution, cyclization, rearrangement, oxidation etc.; accordingly, many thousands of terpenes have been described in the literature. Nitrogen-containing compounds originating from the terpenes have also been reported, but these are counted among the alkaloids (e.g. the Gentiana alkaloids) [Rxc3x6mpp Chemie Lexikon [Rxc3x6mpp""s Chemical Encyclopedia], 9th Edition, Volume 6, page 4508 ff., Georg Thieme Verlag, Stuttgart/New York, 1992]. These terpenes, however, differ fundamentally from the memno peptides according to the invention, wherein terpenes do not contain a polyketide moiety with which they can bind nitrogen.
Examples of further, known nitrogen-containing terpenes are:
Stachybocins [J. Antibiotics, 48: 1396 (1995)];
Stachybotrins [Y. Nozawa et al. J. Antibiotics, 50: 635-645 (1997)];
Spirodihydrobenzofuran lactams[J. Antibiotics, 49: 13 (1996)];
Nakijiquinones [Tetrahedron, 51: 10867-10874 (1995)];
F1839-A to J are nitrogen-containing terpenes having polyketide moieties [Japanese Patent 061864133 and 08283118]. They are cholesterol esterase inhibitors.
These terpene derivatives were synthesized from various strains of the genera Memnoniella echinata and Stachybotrys and others. They were described as antagonists of the endothelin receptor, as inhibitors of HIV-1 protease and of cholesterol esterase and as hair tonics. The inositol monophosphatase inhibitor L-671,776 was moreover isolated from cultures of the strain Memnoniella echinata, ATCC 20928 [Y. K. T. Lam et al. J. Antibiotics, 45, pp.1397-1402, (1992)].
The memno peptides according to the invention have a differing spectrum of activity. A conspicuous feature is their activating effect on SERCA2 and thus on the insufficient heart.
The present invention thus relates to compounds of formula (I) 
wherein
R1 and R2 together are double bonded O, or H2, or H and OH, or H and Oxe2x80x94C1xe2x80x94C4-alkyl;
R3 and R4 together are double bonded O, or H2, or H and OH, or H and Oxe2x80x94C1xe2x80x94C4-alkyl;
R8 is chosen from H, OH, C1xe2x80x94C4-alkyl and Oxe2x80x94C1xe2x80x94C4-alkyl, such as O-methyl; R6 is a group of formula (II) 
wherein R9 is H or a glycosidically bonded sugar, or
a group of the formula (III) 
wherein R10 is H or a glycosidically bonded sugar,
and wherein,
if R6 is a group of the formula (II), then R5 is a bond to the carbon atom C9 of the formula (II) and R7 is H, or R7 is a bond to the carbon atom C9 of the formula (II) and
R5 is H, and
if R6 is a group of the formula III, then R5 and R7 are H;
A is an amino acid,
n is an integer chosen from 1 to 12, wherein each A is the same or different from every other A,
wherein the nitrogen atom in the isoindole ring of formula (I) is the N-terminal amine nitrogen of the first amino acid of the (A)n group;
or a salt or derivative thereof;
with the proviso that
A is an amino acid other than Glu when n is 1, and R1 and R2 together are double bonded O, and R3 and R4 together are H2, and R6 is a group of formula (II), and R5 is a bond to the carbon atom C9 of the group of formula (II), and R7 is H, and R8 is H and R9 is H.
In formula (I), (A)n can be at least one natural amino acid selected from: Gly, Ala, Val, Leu, Ile, Pro, Ser, Thr, Phe, Tyr, Trp, Lys, Arg, Asp, His, Glu, Asn, Gln, Cys and Met. (A)n can be a peptide chain having from 2 to 12 amino acids. In one embodiment (A)n comprises 10 amino acids.
C1-C4-Alkyl is a straight-chain or branched alkyl having 1 to 4 carbon atoms, such as methyl, ethyl, i-propyl and tert-butyl.
The sugar can be a hexose, for example an aldohexose, such as mannose, glucose or galactose, which may be optionally substituted with additional groups, such as C1 to C4-Alkyl or NH2.
The present invention furthermore relates to all obvious derivatives of the compounds of the formula I. Derivatives are salts, reduction products, esters, ethers, acetals as well as amides and N-alkylation products, moreover all optical antipodes, diastereomers and all stereomeric forms.
In one embodiment, the compound of formula (I) has formula (IV) 
where the groups R1, R2, R3, R4, R9 and (A)n have the meaning as indicated above.
In the formula (IV), R1 and R2 taken together can be double bonded O, R3 and R4 together can be H2 and R9 can be H.
In one embodiment, the inventive compound is memno peptide A, C76H108N16O18S, of the formula (IVa) 
In this formula, the decalin structure having the 4-methyl and a methylene group is the terpene moiety; the substituted isoindole ring is the ketide moiety. The amino acid sequence:
Met His Gln Pro His Gln Pro Leu Pro Pro (SEQ ID NO:1) is a part of the casein sequence. The methionine (Met) can be oxidized to the sulfoxide.
Formula (IVb) shows a preferred spatial form: 
wherein (A)n has the meaning as indicated above.
The physicochemical and spectroscopic properties of the preferred compound according to the invention can be summarized as follows:
Memno peptide A
Appearance: colorless substance soluble in polar organic solvents and in water. It is stable in neutral, mildly acidic medium.
Empirical formula: C76H108N16O18S,
Molecular weight: 1565.87 Da,
UV absorption (xcexmax): 270 nm,
NMR data: see Table 1
The numbering of the carbon atoms and the associated NMR chemical shifts are classified according to the numbering procedure for cyclic sesquiterpenes.
FIG. 1: Numbering of a cyclic sesquiterpene ketolide.